The long-term goal of the project is to understand the mechanism of endocytosis of growth factor receptors, using endocytosis of the receptor for epidermal growth factor (EGF) as an experimental system. EGF receptor is an important regulator of cell proliferation in many tissues and is implicated in the development of several types of tumors. EGF binding to the receptor leads to activation of the receptor tyrosine kinase and causes rapid endocytosis and subsequent degradation of EGF:receptor complexes in lysosomes. Ligand-induced down-regulation of the receptor resulting from accelerated endocytosis is a specific phenomenon for all growth factor receptors with intrinsic tyrosine kinase activity. Endocytosis of growth factor receptors is a major regulator of the amount and subcellular distribution of activated receptors in the cell. Therefore, endocytosis may determine the intensity and cellular targeting of mitogenic and transforming signalling by growth factor receptors. However, the mechanism of the specific steps of growth factor receptor endocytosis is not yet understood. Proposed research will focus on the analysis of EGF receptor internalization which is the step determining the rate of receptor down- regulation. The emphasis of the project will be on the search for and characterization of adaptor molecules that allow effective interaction of the receptor with a plasma membrane coated pit, a key element of endocytic apparatus. The general strategy of these studies splits into two parallel and related directions. The major project is whether direct binding of EGF receptors to plasma membrane adaptor proteins occurs, and which adaptor protein functions as a receptor-binding site. The involvement of additional unidentified proteins in the internalization of the EGF receptor will be also investigated. This project will be accompanied by the analysis of the kinetic characteristics of EGF receptor internalization. Identification of adaptor proteins should provide basis for understanding the mechanisms of saturability and specificity of EGF receptor endocytosis. Proposed studies of the dynamics of internalization may, in turn, help to identify proteins regulating the endocytosis of EGF receptors and understand the mechanism of this regulation. Experiments in the first stages of the project will be based mainly on biochemical methods and should result in identification of adaptor proteins important for the EGF receptor endocytosis. Further development of the project will include an extensive application of molecular biology methods in order to clone and characterize a novel protein(s) and to define the structure of the receptor binding domains within the adaptor molecule.